The elementary steps of DNA polymerization catalyzed by T7 DNA polymerase have been resolved by transient-state analysis of single nucleotide incorporation, leading to the complete pathway: where Ε, D, Ν, and Ρ represent T7 DNA polymerase, DNA primer/template, deoxynucleoside triphosphate, and inorganic pyrophosphate, respectively. A DNA primer/template consisting of a synthetic 25/36-mer has been used as a substrate for correct nucleotide incorporation of dTTP in all the experiments. The rate constants and equilibrium constants of each step have been established by direct measurement of individual reactions and fit by computer simulation of the data to obtain a single set of rate constants accounting for all the data. Analysis of the single-turnover kinetics provided measurements of equilibrium dissociation constants for 25/36-mer, dTTP, and PPi equal to 18 nM (k−1/k1), 18 μΜ (k−1/k1), and 2 mM (k5/k−5), respectively. The rate-limiting step during single-nucleotide incorporation has been identified as a conformational change, E·Dn·N → E′·Dn·N, which occurs at a rate of 300 s−1 (k2) upon binding of the correct dNTP. Accordingly, tighter binding of the transition states for the reaction resulting from the conformational change facilitates the phosphodiester bond formation. The chemical step itself was excluded as the rate-limiting step because of the small phosphothioate elemental effect. An observed rate constant of 70 s−1 for dTTP(αS) incorporation suggest that the chemical step (k3) occurs at a fast rate, ≥9000 s−1. Following chemistry, the resulting ternary complex, E′·Dn+1·P, undergoes a second conformational change at a rate of 1200 s−1 (k4), leading to release of PPi and translocation of the DNA to continue subsequent cycles of polymerization. The rate constants of the reverse steps, 100 s−1 (k2), ≥18 000 s−1 (k−3) and 18 s−1 (k−4), were derived as fits to the data based upon simulation of single-turnover kinetics of pyrophosphorolysis including measurements of pyrophosphate exchange and the overall equilibrium constant of 1.0 × 104 for elongation of E·25/36-mer to E·26/36-mer and analysis of the kinetics of the pulse-chase experiment. These studies provide the first complete and self-consistent thermodynamic descriptions of DNA polymerase and establish the basis for quantitative assessment of the reactions contributing to its extraordinary fidelity. The rate constants of the reverse reaction (pyrophosphorolysis) could only be measured by using an exonuclease-deficient mutant of gene 5 protein because of the high exonuclease activity of the wild-type enzyme. The double mutation D5A,E7A resulted in complete inactivation of the exonuclease activity. The mutant was fully characterized, and all rate constants for the polymerization reaction were shown to be identical with those of the wild-type enzyme. Its excision rate for single-stranded DNA was shown to be reduced by a factor of 106, while its excision rate for double-stranded DNA was not measurable.
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